Determining how cell sense and adapt to fluctuating O2 levels in their environment is a fundamental problem in biology. Our studies focus on the regulatory strategies used by the facultative anaerobe E. coli to respond to O2 availability. These studies have led us to investigate two Fe-S cluster transcription factors, FNR and IscR. A picture is emerging that utilization of Fe-S proteins as sensors of O2 and other biologically relevant oxidants has taken advantage of the special properties of these metal centers and involves both regulation of cluster destruction and/or cluster synthesis. Thus, our studies should advance our understanding of how Fe-S clusters function as sensors of O2 and other important oxidants. The global regulator FNR controls transcription of genes under anaerobic growth conditions and serves as a paradigm for sensing cellular O2 levels. FNR is inactivated by cluster destruction upon the O2 dependent removal of its [4Fe-4S]2+ cluster that is required to maintain an active conformation. In this grant period, the fate of apoFNR that is produced from this reaction will be studied in order to develop a comprehensive model for how FNR is regulated at the cellular level. We will test whether apoFNR is the form of FNR that is proteolyzed by the CIpXP protease under aerobic growth conditions. In addition, we will determine whether apoFNR can be reactivated when O2 becomes limiting. Lastly, structural studies of FNR will be initiated to determine how the Fe-S cluster of FNR alters its conformation. IscR contains a [2Fe-2S] cluster and is a represser of genes encoding functions for Fe-S cluster biogenesis (isc operon). Our hypothesis is that IscR is a sensor of cellular demands for Fe-S cluster biogenesis and it is also involved in O2 regulation of gene expression. The role of the [2Fe-2S] cluster of IscR in sensing the Fe-S cluster status of cells will be investigated. To gain new insights into how these transcription factors reprogram metabolism under aerobic and anaerobic conditions, we will study the regulation of FNR and IscR target genes that we recently identified using a genome-wide approach. In particular, we will investigate the mechanism by which FNR antagonizes IscR function under anaerobic conditions to impart a new strategy for the O2 regulation of gene expression. Our studies should provide important new insights into the conserved regulatory strategies for sensing O2 that are used by a wide variety of bacteria including pathogenic organisms and provide fundamental mechanisms of O2 sensing and Fe-S based sensing that apply to all organisms, which when gone awry can cause human disease.